The long-term goal of this project is to establish a set of powerful technologies that enable special and temporal control of the epigenome. We hypothesize that, by selectively and biochemically perturbing a single protein (or even single domain within a protein), we can control the epigenome with high precision. Thus, this project aims to utilize state-of-the-art protein engineering technologies to generate high-performance binding proteins to epigenetic regulatory proteins that can be genetically encoded for intracellular use. We will utilize the designer binding protein platform, termed monobody that we have pioneered and refined over the last decade. Monobodies are designer binding proteins built using the fibronectin type III (FN3) scaffold. Unlike conventional antibodies and their fragments, FN3 lacks disulfide bonds and thus monobodies fold into their functional form under reducing conditions, such as the nucleus and cytoplasm within cells. Therefore, the monobodies are particularly suited as genetically encoded, intracellular inhibitors against epigenetic regulatory proteins. By utilizing sophisticated technologies that we have developed over the last decade, we will generate monobodies to many epigenetic regulatory proteins that have high affinity and exquisite specificity. We will develop companion technologies that enable novel applications of monobodies toward understanding and controlling epigenetic regulatory processes. Specifically, we propose the following aims: Aim 1. To develop high-specificity, high-affinity monobodies to readers and writers of chromatin modifications. Aim 2. To develop technologies for using monobodies as genetically encoded inhibitors directed to epigenetic regulatory proteins and validate monobodies from Aim 1 for intracellular use. Aim 3. To develop chemoepigenetic technologies for temporal control of the chromatin regulators in developing cells using monobody inhibitors, in particular CHD7 in the context of neural crest differentiation and the CHARGE syndrome. We will make these powerful tools available to the community. We have assembled a team of experts with complementary skills and this project will leverage resources and expertise available through a network of collaborators. We have a strong track record of innovations and enabling the research community. Technologies and knowledge gained in this project will provide the epigenomics research community with novel and powerful tools and lead to new approaches to controlling the epigenome for positively impacting human health. Together, this project is perfectly aligned with the vision of the RFA.